Salt stabilization of antibody-enzyme conjugates heat-dried into paper

ABSTRACT

New and useful methods of producing stabilized enzyme antibody conjugates are disclosed which are particularly useful in forming multi-layer immunoassay test devices. In particular, the invention concerns the formation of a manganese ion and enzyme-antibody conjugate in aqueous solution and drying the solution to produce a dry stabilized enzyme-antibody conjugate. Further, this stabilized enzyme-antibody conjugate can be formed on a continuous web and dried in a heat tunnel. This continuous manufacturing process allows for the more efficient production of multi-layer test strips.

This is a continuation of application Ser. No. 504,116, filed on Apr. 3,1990, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to a method of producing stable immunoassayreagents. More particularly, it relates to a test strip upon which isdeposited stabilized enzyme antibody conjugates. Enzyme-based diagnosticassays are used to test for the presence of antigens or antibodies inhuman body fluids. Many methods are used including the use of liquidreagents, radioimmunoassay methods, the use of dry test strips, and theuse of coated test slides. U.S. Pat. No. 3,876,504 to Koffler teachesthe use of a glass or plastic slide coated with a gelatin and a poroussurface forming material to which is bonded either component of theantibody-antigen reaction of interest in an insolubilized form. Theslide is air dried and desiccated. The other component, either antigenor antibody, is conjugated with an enzyme, and this conjugate is mixedwith the body fluid to be applied to the slide. A coloring agentactivatible by the enzyme is finally applied to the test slide in orderto provide an indication of antibody-antigen reaction.

The reagents and indicators for immunoassay are fragile and oftendeteriorate in normal storage operation conditions. Several methods havebeen attempted to achieve a more stabilized reagent. U.S. Pat. No.3,860,484 to O'Malley discloses a process of stabilization ofunconjugated enzymes by contacting them with synthetic polymers andcopolymers. The resulting solution is then freeze dried to form a dryproduct.

U.S. Pat. No. 4,806,343 to Carpenter et al discloses a method forpreserving the activity of proteins after freezing by exposing theprotein to a carbohydrate and a transition metal ion and then freezingthe protein. Useful metal ions discussed include divalent ions of Zn,Cu, Cd, Ni, and Co.

U.S. Pat. No. 4,563,425 to Yoshioka et al discloses a method to inhibitenzyme deactivation when the enzyme is contacted with a glucose basedsubstrate solution. This method involves the addition of carrier boundmetal ions to the substrate solution before termination of the enzymesubstrate reaction. The metal ion may be one or more of Ti, V, Cr, Mn,Fe, Co, Cu, Sb, Ce, or Ag. The metal salt may be an acid salt or acomplex salt, such as a halogen salt. The carrier may be, for example, apolysaccharide, polyamide, glass, or ion exchanger.

U.S. Pat. No. 4,024,000 to Shibata et al discloses a method for thestabilization of an aqueous solution of unconjugated beta-amylase enzymeduring purification by adding a divalent or trivalent metal ion to thesolution. The resulting solution is then purified by membrane separationat 45°-55° C. After separation, the concentrated enzyme solution may besalted out by addition of sodium chloride, in order to precipitate abeta-amylase fraction, which may then be dried.

U.S. Pat. Nos. 4,331,761; 4,169,012; and 4,228,240 to Dawson et aldisclose a method for stabilizing aqueous peroxidase containingcompositions by the addition of polyvalent metal ions to thecompositions. Such ions may include Mg, Ca, Sc, Ti, V, Cr, Mn, Fe, Co,Ni, Cu, Zn, Ga, and Al. The metal salts may be sulfates, phosphates,halides, or nitrates. The products of the various Dawson et al patentsmay be either dried or in aqueous solutions. The dried products aredisclosed to be dried from aqueous compositions which then may or maynot be lyophilized. However, the compositions are usually firstlyophilized.

U.S. Pat. No. 4,757,016 to Klenner et al discloses a process forstabilization of a peroxidase enzyme which may be conjugated to anantibody. This is done by incubation of the enzyme with aminopyrine forone hour at 20°-25° C.

U.S. Pat. No. 4,233,405 to Neubeck discloses a spray drying process forpreparation of unconjugated enzyme products. Neubeck discloses theconcentration of a liquid enzyme solution by ultrafiltration and theaddition of water-insoluble salts to the concentrate prior to the spraydrying.

Finally, U.S. Pat. No. 4,446,232 to Liotta discloses dry, layered teststrips suitable for use in enzyme-linked immunoabsorbant assays (ELISA),having a layer of porous material such as nitrocellulose or DBM paperwithin which soluble enzyme-linked antibodies are dispersed. However,the only method disclosed by Liotta for application of the enzyme-linkedantibodies to the test strip is lyophilization.

Therefore, what is needed is a high temperature stable enzyme-antibodyconjugate. What is also needed is a continuous method to produce storagestable dry, test strip layers suitable for use in ELISA.

SUMMARY OF THE INVENTION

The present invention relates to a method for stabilizing the activityof a dry enzyme-antibody conjugate comprising forming an aqueoussolution of an enzyme-antibody conjugate and a source of manganese ionand drying the solution at an elevated temperature and ambient pressureto form a manganese ion stabilized conjugate. In another embodiment, thepresent invention relates to a continuous method for the manufacture ofa stabilized, dry test strip for immunoassay comprising forming anaqueous solution of an enzyme-antibody conjugate and a source of amanganese ion, contacting a continuous web with the solution and dryingthe web at elevated temperatures and ambient pressure. The term"stability of enzyme activity" as used herein the specification and theclaims means increasing the activity of the stored enzyme significantlyabove that of an untreated enzyme. The term "antibody stability" as usedherein the specification and the claims means increasing the antibodybinding activity significantly above that of an untreated antibody. Theterm "conjugate" as used herein the specification and the claims means aphysical coupling, e.g., covalent binding, of the antibody and enzyme.The phrase "drying at elevated temperatures" as used herein thespecification and the claims means drying of a substance at temperaturesgreater than room temperature.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

I have found that it is possible to stabilize an enzyme-antibodyconjugate by adding manganese ions in an aqueous solution and thermallydrying the solution. This is preferably accomplished through theaddition of a manganese ion source to an aqueous enzyme-antibodysolution. Preferably, enough metal ion source is added to form anaqueous solution of up to about 1M of manganese ion. More preferably,the manganese source is added to solution at about 50 mM to about 500mM. However, the order of addition is not critical. The enzyme-antibodymay also be added to a manganese ion source containing solution. Themetal ion source is preferably introduced as a salt; sulfates andhalogens are more preferred, and chlorides are most preferred.

This invention may be used to stabilize enzyme-antibody conjugatesgenerally. More preferably, the enzyme-antibody conjugate stabilized isan antibody conjugated with horseradish peroxidase (HRP), and mostpreferably, HRP is conjugated with an antibody specific fortheophylline. The enzyme-antibody conjugate may be prepared by any meansknown by those skilled in the art. One method is the standard periodatemethod of Wilson and Nakame mentioned in the Liotta patent.

Once the manganese ion source and conjugate have been mixed in theaqueous solution, the solution is introduced to elevated temperaturesfor drying. Preferably, the drying is done at temperatures from about40° C. to about 100° C. More preferably, the drying is performed betweenabout 55° C. and about 80° C. Convection, conventional, infrared, heattunnel, and microwave ovens are preferred as sources of elevatedtemperatures. More preferably, a heat tunnel is used. Most preferably, aheat tunnel with a plurality of heating zones is used.

The manganese ion stabilized enzyme-antibody conjugate may be dried foruse in any manner. It is preferred that the conjugate is dried onto asubstrate. More preferably, the substrate is paper, glass, fiberglass,agarose, nitrocellulose, polymeric materials, or mixtures thereof. Mostpreferably, the substrate is paper, nylon, dacron, fiberglass, agarose,nitrocellulose, polyacrylamide, or mixtures thereof. This substrate ispreferred in a woven, non-woven, or a sheet form. Most preferably, thestabilized conjugate is dried onto a substrate and incorporated into aLiotta-type test strip as disclosed in U.S. Pat. No. 4,446,232 describedabove and incorporated herein by reference.

The invention is preferably practiced by forming the enzyme-antibodyconjugate/manganese ion aqueous solution and introducing into thesolution a continuous web of a substrate, allowing the solution tosaturate the substrate. The saturated substrate is then removed from thesolution and drawn through a heat tunnel to dry. The heat tunnelpreferably has a plurality of zones of differing heat levels. The firstzone is preferably the hottest and the last zone is preferably thecoolest. Upon removal from the heat tunnel, the continuous web ispreferably cut into several smaller pieces of test material. These testpieces are most preferably incorporated into a Liotta-type strip.

EXAMPLES

The present invention is demonstrated by the following examples. Theseare merely illustrative; obvious modifications can be carried out inlight of the previous discussion and appended claims. All percentagesand proportions referred to in this description are by weight unlessotherwise indicated.

In the Examples, the following material and methods were used.

A. The enzyme-antibody conjugate:

A fragment of a monoclonial antibody specific for theophylline wasconjugated to horseradish peroxidase (HRP). This was purified and keptat -80° C. as a conjugate concentrate until used.

B. Base formulation for impregnation into paper:

HEPES buffer, 100 mM, pH 7.

Ovalbumin, 1%

Mannitol, 2.5%

Rabbit antitheophylline antiserum (See application Ser. No. 07/284,099)

C. Drying parameters:

Dryer: 3 zones, 18 feet long

Temperature:

zone 1=175° F.

zone 2=160° F.

zone 3=130° F.

Web speed: 2 feet per minute

Air Pressure: 3 inches of water.

D. Enzyme survival assay:

The conversion of 3,3',5,5'-tetramethylbenzidine (TMB) to a coloredproduct was expressed as the percent enzyme activity remaining of theliquid used to impregnate the paper matrix (Miles Technical Bulletin,1983 and Bos, E. S., et al, 1981, J. Immunoassay 1:187.)

E. Performance survival assay:

The production of a given change in color, as percent reflectance (% R)between 0-40 micrograms/milliliter of theophylline on three layerstrips, was expressed as the percent of the performance remaining of theliquid used to impregnate the paper matrix. Performance requires afunctional antibody portion of the enzyme-antibody conjugate as well asthe non-conjugated antibody added to the formulation.

EXAMPLE I

A series of test runs was made using the base formulation, addingvarious salts to compare the effect on the recovery of HRP activityfollowing the drying process (termed Loss on Processing, LOP) measuredin terms of the percentage of a liquid control (% LC).

    ______________________________________                                                           Concentration                                                                              % LC                                          Formulation                                                                             Salt     (mM)         Enzyme (LOP)                                  ______________________________________                                        1         None     --           63.8                                          2         EDTA      10          77.4                                          3         KCl      100          69.8                                          4         NaCl     100          70.5                                          5         MnCl.sub.2                                                                             100          84.8                                          6         MnCl.sub.2                                                                             200          96.9                                          ______________________________________                                    

From the data, it is apparent that MnCl₂ performs significantly betterthan monovalent salts and sequestering agents, and it performs muchbetter than no salt stabilizers at all.

EXAMPLE II

A series of tests was conducted using the base formula and addingvarious metal salts. The following table shows the effect of these saltson enzyme and antibody survival after drying during storage for one weekat 60° C. (termed Loss on Stress, LOS) measured in terms of % LC.

    ______________________________________                                                                            % LC                                                                          Heat Stress                                                Concentration                                                                             Enzyme Performance                               Formulation                                                                            Salt    (mM)        (LOP)  (LOS)                                     ______________________________________                                        7        None    --          23.2   28.0                                      8        NaCl    100         36.6   56.8                                      9        MnCl.sub.2                                                                            100         59.8   60.3                                      10       MnCl.sub.2                                                                            200         92.8   95.3                                      ______________________________________                                    

Again, MnCl₂ performs significantly better than monovalent salts andmuch better than no salt stabilizer at all.

What is claimed is:
 1. A method for stabilizing the activity of a dry enzyme-antibody conjugate comprising:(a) forming an aqueous solution comprising(i) a peroxidase-antibody conjugate, and (ii) a source of manganese metal ion in an amount sufficient for stabilizing the catalytic and binding activity of said conjugate; and (b) drying the solution at a temperature between 40°-100° C. form a manganese ion conjugate in which both the catalytic and binding activity of the peroxidase antibody conjugate is stabilized.
 2. The method of claim 1 in which the source of the manganese ion is present in a concentration up to about 1M.
 3. The method of claim 2 in which the source of the manganese ion is present in a concentration of from about 50 mM to about 500 mM.
 4. The method of claim 1 in which the drying temperature is from about 55° C. to about 80° C.
 5. The method of claim 1 in which the peroxidase-antibody conjugate comprises a horseradish peroxidase-antibody conjugate.
 6. The method of claim 5 in which the horseradish peroxidase-antibody conjugate is a conjugate of horseradish peroxidase and the antibody specific to theophylline. 